By the way of background, for many years wall "loosening enzymes" have been implicated in the control of plant cell enlargement (growth), largely on the basis of rapid biophysical and biochemical changes in the wall during auxin-induced growth (reviewed by Cleland and Rayle, Bot. Mag. Tokyo 1:125-139, 1978; Taiz, Annu. Rev. Plant Physiol., 35:585-657, 1984). Plant walls contain numerous hydrolytic enzymes, which have been viewed as catalysts capable of weakening the wall to permit turgor-driven expansion (reviewed by Fry, Physiol. Plantarum 75:532-536, 1989). In support of this hypothesis, Huber and Nevins (Physiol. Plant. 53: 533-539, 1981) and Inoue and Nevins (Plant Physiol. 96:426-431, 1991) found that antibodies raised against wall proteins could inhibit both auxin-induccd growth and wall autolysis of corn coleoptiles. In addition, isolated walls from many species extend irreversibly when placed under tension in acid conditions in a manner consistent with an enzyme-mediated process (Cosgrove D. J. Planta 177:121-130, 1989). Despite these results and other evidence in favor of "wall-loosening" enzymes, a crucial prediction of this hypothesis has never been demonstrated, namely that exogenously added enzymes or enzyme mixtures can induce extension of isolated walls. To the contrary, Ruesink (Planta, 89:95-107, 1969) reported that exogenous wall hydrolytic enzymes could mechanically weaken the wall without stimulating expansion. Similarly, autolysis of walls during fruit ripening does not lead to cell expansion. Thus a major piece of evidence in favor of wall-loosening enzymes as agents of growth control has been lacking.
The walls of growing cucumber seedlings possess extractable proteins which can induce extension of isolated walls. Additionally, we identified two specific wall-associated proteins with this activity. We proposed the name "expansin" for this class of proteins, defined as endogenous cell wall proteins which restore extension activity to inactivated walls held under tension. We further propose the specific names "expansin-29" and "expansin-30" (abbreviated Ex-29 and Ex-30, with respect to their relative molecular masses; McQueen-Mason et al. Plant Cell, 4:1425-1433, 1992) for the two proteins isolated from cucumber. More recently we have identified an oat coleoptile wall protein that induced extension in isolated dicot walls (Z. C. Lee et al., 1993, Planta, 191:349-356). The oat protein has an apparent molecular mass of 29 kD as revealed by SDS-PAGE. For clarity we will refer to the cucumber proteins as cEx, and to the oat proteins oEx. New data demonstrate that an expansin-like protein may be found in proteins obtained from the digestive track of snail and its feces. We will refer to the snail protein as sEx.
During studies of the biochemical mechanism of action of expansins, we found that they had the ability to weaken the hydrogen bonding between plant cell wall polysaccharides (such as cellulose fibers). These findings allow us to propose the following commercial uses of these novel proteins, including paper treatment, agricultural uses, and a variety of use in food and industrial markets.
The paper products industry employs 3/4 million workers and is a $60-billion industry in the U.S. alone (plus $40 billion in retail sales). Recycling is a growing concern and will prove more important as the nation's landfill sites become more scarce and more expensive. According to 1992 testimony before congress on the problems and opportunities of paper recycling, the need for improved technology in paper recycling is urgent and of high priority. The use of expansins in this industry may be well received at this time.
Paper derives its mechanical strength from hydrogen bonding between paper fibers, which are composed primarily of cellulose. During paper recycling, the hydrogen bonding between paper fibers is disrupted by chemical and mechanical means prior to reforming new paper products. Expansins may be used to weaken the hydrogen bonding between the paper fibers of recycled paper. As demonstrated in this invention disclosure, expansins, at very low concentrations, in fact weaken commercial papers, including slick paper from magazines and catalogs. These latter types of papers are difficult to recycle because they are not easily disrupted in commercial recycling processes.
The advantages of using expansins for paper recycling include the following: the protein is nontoxic and environmentally innocuous; it could substitute for current harsh chemical treatments which are environmentally noxious. The protein is effective on paper products which are now recalcitrant to current recycling processes. Its use could expand the range of recyclable papers. Because the protein acts at moderate temperature and in mild chemical environments, degradation of paper fibers during recycling should be reduced. This should allow for recycled paper fibers with stronger mechanical properties and with the ability to be recycled more often than is currently practical. Moreover, savings in energy costs associated with heating and beating the paper may be realized.
Other modes of application of expansins includes production of virgin paper. Pulp for virgin paper is made by disrupting the bonding between plant fibers. Following the reasoning listed above, expansins may be useful in the production of paper pulp from plant tissues. Use of expansins could substitute for harsher chemicals now in usc and thereby reduce the financial and environmental costs associated with disposing of these harsh chemicals. The use of expansins could also result in higher quality plant fibers because they would be less degraded than fibers currently obtained by harsher treatments.
Cellulose is the major structural component of plant fibers used in textiles, paper and rope making. Commercially, cellulose is used as a starting "feed stock" material for the production of many cellulose derivatives, such as cellulose acetate, cellulose nitrate, and carboxymethyl cellulose. It has numerous uses in many industries, including: as a fiber in the paper/pulp and textile industries, as a starting material for manufacture of films, coatings, membranes, thickeners, and as a biomass source for alcohol production. Processing cellulose for these commercial applications is arduous because cellulose is arranged in a microcrystalline lattice structure and incorporated into microfibrils that are mechanically and chemically resistant to chemical, enzymatic and biological degradation.
Native cellulose is found as an ordered microfibril, in which individual glucans laterally associate with one another into tight arrays, or long crystalline ribbons. This arrangement limits access of enzymes or chemicals to the individual glucans. Glucan accessibility is thus one of the major limitations to efficient enzymatic or chemical modification of cellulose.
In order to make the glucans accessible to standard enzymatic or chemical processing, the individual glucans need to be stripped off the surface of the crystalline microfibrils (Bayer, Chanzy, Lamed, and Shoham, 1998). The chemical processes for solubilizing and degrading cellulose for commercial applications generally involve the use of harsh chemicals and extreme conditions. These processes often result in unwanted modification and derivatization of the cellulose. Numerous cellulases are commercially available, however they lack effectiveness because the enzymes cannot gain ready access to the crystalline polymer.
If a means could be found to break open, decrystallize, or by another enzymatic mediated process to modify cellulose under milder conditions, this might have considerable practical benefit to industries that process cellulose or manufacture products utilizing cellulose.